The p38 MAPK pathway was initially identified as a mediator of inflammatory and stress responses. We and others have shown that this pathway also participates in tumor suppressing cellular responses such as oncogene-induced senescence. Oncogene-induced senescence is an irreversible form of proliferative arrest triggered upon activation of oncogenes in normal cells, which is morphologically identical to replicative senescence resulted from cellular aging. Studies in the current funding period of this grant, aimed to delineate the p38 pathway components involved in senescence, demonstrates that p38 acts through a downstream substrate kinase PRAK. Further studies reveal that p38/PRAK-mediated senescence operates in vivo to suppress cancer development in murine models, and that PRAK is a tumor suppressor protein that is inactivated in certain types of human cancer. Four p38 isoforms (p381, 2, 3 and 4) exist in mammals, each encoded by a different gene. We found that these p38 isoforms play fundamentally different roles in oncogene- induced senescence. They differ both in their ability to mediate senescence induction and in the mechanism by which they mediate senescence. While p381, 3 and 4 are essential for oncogenic ras-induced senescence, p382 is dispensable. Among the isoforms that are crucial, p383 contributes to oncogene-induced senescence by stimulating the transcriptional activity of p53 via phosphorylation of p53 at Ser33. In contrast, p381 mediates senescence induction through a p53-indepenedent mechanism, by increasing the expression of p16INK4A. In this renewal application, we seek to further delineate the mechanism underlying the differential roles of the p38 isoforms in oncogene-induced senescence, and to determine the biological relevance of senescence induction mediated by the p38 isoforms in cancer development. First, we will investigate the function of p381 in senescence, by analyzing the mechanism by which p381 mediates the ras-induced expression of p16INK4A and the functional consequence of this regulation. Next, we will characterize the tumor suppressing activity of the p38 isoforms in cell culture by testing the effect of inactivation of the p38 isoforms on the tumorigenic phenotypes of transformed model cell lines with defined genetic alterations. Finally, we will determine the role of the p38 isoforms in oncogene-induced senescence and tumor suppression in vivo in a murine skin carcinogenesis model. Results from the proposed work will provide mechanistic insights into the differential roles of the p38 isoforms in oncogene-induced senescence, and define the function of the p38 isoforms in mediating oncogene-induced senescence and tumor suppressing in vivo. Moreover, since pharmaceutical inhibitors of p38 are currently under development as anti-inflammatory drugs, the proposed studies will help improve the safety of these drugs by identifying the tumor-suppressing p38 isoforms that should be excluded as drug targets. PUBLIC HEALTH RELEVANCE: The overall goal of this application is to delineate the function of the p38 isoforms in oncogene-induced senescence, a fail-safe tumor suppressing cellular response. The proposed work will advance our understanding of the mechanisms that protect human being against cancer. Since p38 inhibitors are currently pursued as anti-inflammatory drugs, results from the proposed studies will help guide the design of safe drugs without cancer-promoting side effects, by identifying the tumor-suppressing p38 isoforms that should be avoided as drug targets.